PROJECT SUMMARY The overall goal of this project is to develop and compare three new HIV envelope (Env) trimer delivery vaccines that are designed to mimic the natural presentation of the transmembrane glycoprotein complex (the spike) that occurs during HIV infection. Our vaccine design approach emphasizes delivery of authentic Env trimer primarily because antibodies (Abs) with broad HIV-neutralizing activity (bNAbs) have been elicited only in patients infected with HIV. Recently, several new Env glycoprotein designs have been developed that should enable improved presentation of the native trimeric Env. These new immunogens assemble into correctly folded trimeric complexes that are significantly more stable, which should make it possible to stimulate B cells with a more uniform well-ordered mimic of the functional Env trimer. An additional challenge to realizing the potential benefits of the improved immunogen will be identification and development of vaccine delivery platforms that can present the correctly-configured glycoprotein complex in vivo and also stimulate immune system functions required to drive the B cell response beyond development of antibodies with limited neutralization breadth. Accordingly, the goal of this research project is to apply this promising Env trimer stabilization technology to the development of three novel vaccines that will present nearly identical stable, well-ordered viral spikes, but use substantially different delivery mechanisms that will stimulate distinctive profiles of immune system functions. This will make it possible to conduct comparative studies to specifically analyze how vaccine platforms and their unique immunostimulatory properties affect the qualities of the Env antibody response. Each vaccine will deliver a stabilized HIV clade C strain 1086 (C.1086) Env immunogen that will be developed using the new native flexibly-linked (NFL) Env trimer technology (EnvNFL), which will be adapted for expression of transmembrane glycoproteins. The three Specific Aims for the project are focused on development and evaluation of the new vaccines and production of vaccine material needed to support preclinical testing in animal models, which will be conducted in collaboration with the Research Project Teams and Technical Cores that are members of this HIVRAD Program. This Project Team will develop novel stabilized spike delivery vaccines based on three immunogenic vaccine platforms, including: 1) an innovative mRNA vector and adjuvant delivery system called RNActive; 2) EnvNFL trimers arrayed on inactivated vesicular stomatitis virus (VSV) particles formulated with adjuvant; and 3) EnvNFL delivered with a live replication-competent VSV vector that incorporates Env in its viral envelope. The Project Team will generate and optimize the three novel vaccine candidates, ensure that they effectively deliver stabilized transmembrane spikes with the expected antigenicity profiles, and provide well-characterized, high-quality vaccine material for evaluation in preclinical immunogenicity and efficacy studies.